Protein phosphorylation regulates numerous cellular functions including ion channel function. Among the largest ion channels are the calcium release channel/ryanodine receptors, which in cardiac and skeletal muscle are required for excitation contraction coupling. Hyperphosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) causes defective function of the channel in human heart failure. Altered calcium handling in the heart due to hyperphosphorylation of RyR may account for, in part, abnormal systolic and diastolic function and arrhythmogenesis. RyR2 macromolecular complex formation involves the recruitment of adaptor proteins/kinases and phosphatases to the ion channel. The local targeting of these signaling molecules is through the specific binding of adaptor proteins to the channel via leucine/isoleucine zipper domains. We have recently found that RyR2 forms a macromolecular complex with mAKAP-PKA and two phosphatases (PP1 through the adaptor protein, spinophilin and PP2A through an unknown adaptor protein). The objectives of this project are to examine the mechanisms that underlie the recruitment of adaptor proteins and kinases/phosphatases to cardiac ion channels. Since several cardiac ion channels (RyR2, a1c subunit of L-type Ca2+ channel, KvLQT1) have leucine zipper motifs and are regulated by phosphorylation, understanding the mechanism(s) of specific modulator targeting via leucine zippers may lead to a new paradigm for the study of ion channel regulation. Three specific aims are proposed to elucidate the targeting and functional role of RyR2-associated phosphatases: (1) Characterization of the mechanism by which PP1/spinophilin interacts with RyR2; (2) Characterization of the leucine zipper motif and identification of the targeting adaptor protein responsible for PP2A/RyR2 interaction; and (3) Functional characterization of the role of the anchoring of phosphatases to RyR2 using expression of constructs that specifically disrupt local signaling. These findings may lead to novel and specific therapies to prevent heart failure and arrhythmogenesis. We plan to utilize site-directed mutagenesis to further understand the targeting of adaptor proteins/phosphatases to RyR2 and to determine whether the association of and function of phosphatases are regulated in disease states. We will utilize novel strategies based upon our findings of the binding sites of these regulatory proteins to test whether targeting of PP1 and/or PP2A to RyR is (are) responsible for the dephosphorylation of the channel. We anticipate that the proposed studies of the intracellular calcium release channel, will provide new information that could lead to the development of novel pharmacological approaches to the treatment of heart failure and arrhythmogenesis and may serve as a model for the study of other cardiac ion channels.